The glycoprotein isolated from vesicular stomatitis virus is mitogenic for mouse B lymphocytes.

The glycoprotein (G protein) of VSV was purified from the intact virion by Triton X-100 extraction. The isolated G protein has been shown to be a T cell-independent, B lymphocyte mitogen and polyclonal activator. Neither G protein nor the intact virion are stimulatory for murine T lymphocytes. The greater the density of G protein in lipid vesicles or the degree of aggregation of isolated G protein, the more highly stimulatory it is for murine splenocytes. As G protein is spread out in artificial vesicles, it becomes less mitogenic. It is probable that other viral components are also stimulatory since the Triton-insoluble pellet and VSV from which the G protein has been enzymatically removed retain mitogenic activity. To out knowledge, this is the first time a purified viral component has been demonstrated to be lymphocyte mitogen.

Vaccination of rhesus monkeys with synthetic peptide in a fusogenic proteoliposome elicits simian immunodeficiency virus-specific CD8+ cytotoxic T lymphocytes.

An effective vaccine against the human immunodeficiency virus should be capable of eliciting both an antibody and a cytotoxic T lymphocyte (CTL) response. However, when viral proteins and peptides are formulated with traditional immunological adjuvants and inoculated via a route acceptable for use in humans, they have not been successful at eliciting virus-specific, major histocompatibility complex (MHC) class I-restricted CTL. We have designed a novel viral subunit vaccine by encapsulating a previously defined synthetic peptide CTL epitope of the simian immunodeficiency virus (SIV) gag protein within a proteoliposome capable of attaching to and fusing with plasma membranes. Upon fusing, the encapsulated contents of this proteoliposome can enter the MHC class I processing pathway through the cytoplasm. In this report, we show that after a single intramuscular vaccination, rhesus monkeys develop a CD8+ cell-mediated, MHC class I-restricted CTL response that recognizes the synthetic peptide immunogen. The induced CTL also demonstrate antiviral immunity by recognizing SIV gag protein endogenously processed by target cells infected with SIV/vaccinia recombinant virus. These results demonstrate that virus-specific, MHC class I-restricted, CD8+ CTL can be elicited by a safe, nonreplicating viral subunit vaccine in a primate model for acquired immune deficiency syndrome. Moreover, the proteoliposome vaccine formation described can include multiple synthetic peptide epitopes, and, thus, offers a simple means of generating antiviral cell-mediated immunity in a genetically heterogeneous population.

An inhibitor of animal cell growth increases cell-to-cell adhesion.

The interactions of both normal and transformed cells with their environment is mediated to a large extent by the cell surface. Succinylated concanavalin A (succinyl-Con A) is a nontoxic and nonagglutinating derivative of the jack-bean lectin concanavalin A. Succinyl-Con A, presumably through an interaction with the cell surface, reversibly inhibits the growth of normal cells and restores a normal growth phenotype to transformed cells. Whereas at high cell densities migration was inhibited, it turned out that at low cell densities where cells are not in contact with each other, cell movement was not affected by succinyl-Con A. Together with some additional observations, this suggests that this lectin derivative increases cell-to-cell adhesion in culture and thereby may influence cell migration. An increase in cell-to-cell adhesion by this lectin derivative may not be brought about simply by physically linking cells together. It occurs after a lag time, possibly by inducing surface changes. The relationship between cell adhesion in culture, cell movement, and cell growth is discussed.

Growth inhibition of transformed cells with succinylated concanavalin A.

Succinylated concanavalin A reversibly inhibits the growth of SV40 transformed mouse 3T3 cells and thus causes an accumulation of the cells in the G1 phase of the cell cycle. In a soft substrate (methylcellulose) succinylated concanavalin A also restores in transformed cells the growth behavior typical of untransformed cells.

Potentiation of DNA mediated gene transfer in NIH3T3 cells by activators of protein kinase C.

The mechanisms involved in the translocation of exogenously added genetic information through the cellular cytoplasm and into the nucleus are essentially unknown. Several trans-cytoplasmic translocation systems operate within cells to transport information received by the plasma membrane into the nucleus. Protein kinase C may be functionally involved in many of these translocation mechanisms. In order to explore the involvement of protein kinase C activation in the cytoplasmic translocation of DNA, NIH3T3 fibroblasts were transfected using the calcium-phosphate co-precipitation method with a plasmid containing the lacZ gene and treated with tetradecanoylphorbol 12,13-acetate (TPA) or 1,2-dioctanoylglycerol (DiC8). Addition of TPA or DiC8 immediately after glycerol shock resulted in a 5-7-fold increase in the number of cells expressing beta-galactosidase as well as a concomitant increase in the total amount of beta-galactosidase activity in the population during periods of transient and stable expression. TPA added at later times resulted in lesser increases in the efficiency of transfection. In contrast, TPA added at the time of addition of the calcium-phosphate precipitate inhibited transfection. In support of a role for protein kinase C activation in enhancing DNA transfection, the TPA analog 4 alpha-phorbol 12,13-didecanoate, which does not activate protein kinase C, was ineffective at enhancing transfection. Furthermore, treatment of cells with the protein kinase C inhibitor sphingosine blocked the TPA-mediated increase in transient and stable expression. The results suggest that protein kinase C activation enhances transfection of exogenous DNA through an as yet unknown mechanism.

Differential effects of the phorbol ester TPA on DNA-mediated transfection in a variety of cell lines.

The efficiency of stable gene transfer and expression in NIH3T3 cells has been shown to be significantly enhanced by a brief treatment with the phorbol ester tetradecanoylphorbol 12,13-acetate (TPA) immediately following calcium-phosphate transfection. Several lines of evidence indicated that this effect was mediated through protein kinase C activation. These studies were expanded to determine whether this was a consistent and widespread phenomenon among other cell lines. The efficiency of transfection in two other established fibroblast lines, LMtk- and 2A3 3T3, was unaffected by TPA treatment, and primary human foreskin fibroblasts were similarly unaffected. Transfection was inhibited by TPA treatment in the transformed cell lines EJ and HeLa. Protein kinase C enzyme assays indicated that TPA causes a translocation of the enzyme from cytosol to membrane in both NIH3T3 and EJ cells, suggesting that the PKC translocation event does not account for the TPA effect on transfection. The TPA-mediated inhibition of transfection in EJ cells was not blocked by sphingosine, suggesting that this phenomenon is unrelated to PKC activation. The results suggest that TPA treatment may either enhance, inhibit, or have no effect on transfection, depending on the cell line.

Chimerasome-mediated gene transfer in vitro and in vivo.

Proteoliposome delivery vesicles can be prepared by the protein-cochleate method [Gould-Fogerite and Mannino, Anal. Biochem. 148 (1985) 15-25; Mannino and Gould-Fogerite, Biotechniques 6 (1988) 682-690]. Proteins which mediate the entry of enveloped viruses into cells are integrated in the lipid bilayer, and materials are encapsulated at high efficiency within the aqueous interior of these vesicles. We describe proteoliposome-mediated delivery of proteins and drugs into entire populations of cells in culture. Material can be delivered gradually by Sendai-virus-glycoprotein-containing proteoliposomes. Alternatively, synchronous delivery to a population can be achieved by exposing cell-bound influenza glycoprotein vesicles briefly to low pH buffer. When DNA is encapsulated, chimeric proteoliposome gene-transfer vesicles (chimerasomes), which mediate high-efficiency gene transfer in vitro and in vivo, are produced. Stable expression of a bovine papilloma virus-based plasmid in tissue-cultured cells, at 100,000 times greater efficiency than Ca.phosphate precipitation of DNA, with respect to the quantity of DNA used, has been achieved. Stable gene transfer and expression in mice has been obtained by subcutaneous injection of chimerasomes containing a plasmid expressing the early region of polyoma virus. In one experimental group, 50% of the mice developed tumors which were shown to express polyoma virus early proteins and contain the transferred DNA. This is the first report of stable gene transfer in animals mediated by a liposome- or proteoliposome-based system.

Liposome mediated gene transfer.

Liposomes, artificial membrane vesicles, are being intensively studied for their usefulness as delivery vehicles in vitro and in vivo. Substantial progress has been made in the development of procedures for liposome preparation, targeting and delivery of contents. The broad flexibility now available in the design of the structure and composition of liposomes, coupled to recent reports of liposome mediated gene transfer in animals, suggest that liposome technology is now poised to be utilized in the creation of custom-designed cell-type-specific gene transfer vehicles.

Lipid matrix-based subunit vaccines: a structure-function approach to oral and parenteral immunization.

Immunization is today the most effective defense mechanism against microbial infections. Although highly effective vaccines are currently available for a number of infectious diseases, vaccine formulations can still be improved in a number of important areas. The ability to induce antigen-specific humoral and cell-mediated immunity is crucial to the development of effective prophylactic and therapeutic vaccines for HIV and other pathogens. The approach of our laboratory has been to design and test simple, highly defined antigen-lipid complexes that would stimulate antibody and cell-mediated immune responses in the absence of any nonspecific immunological activators such as Freund's adjuvant, lipopolysaccharide (LPS), or alum. These studies have provided insight into the relationships between the properties of an immunogen and the induction of the desired immune responses. We have previously utilized this approach to define the minimal structures required for the induction of antibody responses. Our more recent studies have focused on defining the parameters involved in the induction of cell-mediated and mucosal immune responses. Toward this end we have developed a new type of subunit vaccine that is effective when given orally or intramuscularly, and elucidated structure-function relationships in peptide vaccines that affect induction of CD8+ cell responses.

The characteristics of succinylated con A induced growth inhibition of 3T3 cells in tissue culture.

The growth of untransformed 3T3 fibroblasts can be inhibited by dimeric, non-agglutinating concanavalin A prepared by succinylation (Suc-CON A). This growth inhibition is non-toxic, reversible, and specific for Suc-Con A binding; the cell density at which growth terminates is dependent upon the final cell number and independent of the initial (i.e., plating) density. The part of the cell cycle during which Suc-Con A can exert its growth inhibitory effect appears to be restricted to mitosis and/or early G-1 phase.

Lipid matrix-based vaccines for mucosal and systemic immunization.

For more than a decade our laboratories have been combining concepts in biochemistry, virology, and immunology in order to develop a conceptual basis for vaccine design. Our long-term goals have been to construct simple and well-defined immunogens that would stimulate specific immune responses in vivo. Using this approach, we hypothesized that it should be possible to define the structural and biochemical parameters of an immunogen that are necessary and sufficient to stimulate designated effector arms of the immune system. Through the use of covalently coupled peptide complexes, we have been able to define minimal requirements for the induction of humoral immune responses (Mannino et al., 1992). This represents a significant advance in eliciting an immune response to peptides, because it requires only peptides and phospholipids in the absence of additional adjuvants. It is different from the previous use of peptides and liposomes since here the peptides are covalently linked to a hydrophobic anchor and integrated into the phospholipid complex, rather than passively adsorbed or encapsulated. The presentation of peptide as part of a peptide-phospholipid complex (in contrast to encapsulation or nonspecific absorption) may be more similar to the natural presentation of an epitope in the context of an in vivo antigenic challenge. This technology also allows us to incorporate B and Th epitopes in a number of forms--as a single peptide, as two peptides in the same liposome, or as a peptide with viral glycoproteins in the same liposome. These data also demonstrate that Th epitopes do not have to be covalently linked to the B-cell epitope in order to provide help for that epitope. The implications of the data reported here are significant for both basic science and applied technologies. In basic science, the peptide-phospholipid complexes are potentially useful for analyzing the cooperative effects of B- and T-cell epitopes in the in vivo immune response. Since the peptide-phospholipid complexes are totally synthetic and highly immunogenic, they may be constructed in any formulation required to answer questions on the roles of B and T cells in promoting an immune response. Furthermore, since the number of antigenic sites is limited only by the number of peptides included in the peptide-phospholipid complexes, these constructs may be useful in producing antisera or monoclonal antibodies to weakly antigenic regions of a large protein, since the lack of antigenic competition should enhance the immunogenicity of these regions. Clinically, this technology will expand the potential for subunit vaccines.(ABSTRACT TRUNCATED AT 400 WORDS)

Rotary dialysis: its application to the preparation of large liposomes and large proteoliposomes (protein-lipid vesicles) with high encapsulation efficiency and efficient reconstitution of membrane proteins.

An apparatus for rotary dialysis is introduced and described in detail. The component parts are inexpensive, widely available, and relatively easy to modify and assemble. The apparatus achieves increased mixing of the contents of dialysis bags by constant end-over-end rotation. This technique is particularly useful in systems where maximum contact is desired between substances which would tend to partition under standard dialysis conditions. We have applied rotary dialysis to two liposome production methods. These are (i) the calcium-EDTA-chelation method of Papahadjopoulos et al. (1), which produces large unilamellar liposomes from negatively charged phospholipids, and (ii) a procedure for the reconstitution of membrane proteins into liposomes with a large internal aqueous space, which we have developed using the calcium-EDTA-chelation technique as a point of departure. In both techniques, vesicle formation occurs when a calcium-phospholipid precipitate is dissolved by the addition of EDTA. Instead of adding a 150 mM EDTA solution directly, as described in the original method, we have used overnight rotary dialysis against buffer containing 10 mM EDTA at the vesicle formation stage. Materials are encapsulated within the aqueous interior of the vesicles at much higher efficiencies when rotary dialysis is used in either method, compared to efficiencies obtained with direct addition of EDTA (up to 37% of added material vs a maximum published efficiency of 10% for direct addition). Rotary dialysis also promotes the reconstitution of a higher proportion of the membrane proteins present in the dialysis mixture into the bilayer of large liposomes (79 vs 41.6%). It also affects the content of liposomes qualitatively, allowing better reconstitution of the Sendai virus F glycoprotein than does direct addition of EDTA. These effects may be due to the slow time course, the extensive mixing of components, and the low volume-to-phospholipid ratios maintained during vesicle formation.

Aspects of cellular physiology that influence DNA-mediate gene transfer in NIH3T3 cells.

Cellular physiology has a significant influence on the efficiency of various gene transfer procedures, as shown by the fact that transfection efficiency varies dramatically among different cell lines. However, the aspects of cellular physiology which influence the transfection process remain substantially uncharacterized. In this study, NIH3T3 cells were treated with inhibitors of protein synthesis, DNA synthesis, and RNA synthesis to determine the importance of these processes in the calcium-phosphate transfection process. The results suggest that protein synthesis during the first 4 h after DNA addition enhances transfection. In contrast, inhibition of RNA synthesis has no effect on transfection during the first 24 h post-DNA addition. The DNA synthesis inhibitor results remain inconclusive due to a secondary inhibition of an unknown cellular factor. Secondly, agents that destabilize microtubules, microfilaments, and the golgi apparatus were used to determine whether these elements play a role in the transfection process. The results suggest that microtubules are not involved in the transfection process, microfilaments are important but not necessary for the transfection process, and a functional golgi apparatus is essential early in the transfection process. These studies provide a foundation from which further investigations into the cellular processes involved in the uptake and expression of exogenous DNA can proceed.

Antifungal activity of amphotericin B cochleates against Candida albicans infection in a mouse model.

Cochleates are lipid-based supramolecular assemblies composed of natural products, negatively charged phospholipid, and a divalent cation. Cochleates can encapsulate amphotericin B (AmB), an important antifungal drug. AmB cochleates (CAMB) have a unique shape and the ability to target AmB to fungi. The minimal inhibitory concentration and the minimum lethal concentration against Candida albicans are similar to that for desoxycholate AmB (DAMB; Fungizone). In vitro, CAMB induced no hemolysis of human red blood cells at concentrations of as high as 500 microg of AmB/ml, and DAMB was highly hemolytic at 10 microg of AmB/ml. CAMB protect ICR mice infected with C. albicans when the agent is administered intraperitoneally at doses of as low as 0.1 mg/kg/day. In a tissue burden study, CAMB, DAMB, and AmBisome (liposomal AmB; LAMB) were effective in the kidneys, but in the spleen CAMB was more potent than DAMB at 1 mg/kg/day and was equivalent to LAMB at 10 mg/kg/day. In summary, CAMB are highly effective in treating murine candidiasis and compare well with AmBisome and AmB.

Role of intrastructural/intermolecular help in immunization with peptide-phospholipid complexes.

The design of effective subunit vaccines requires the inclusion of both B and T cell epitopes. The best mechanism for including both types of epitopes within an Ag is dependent upon how the Ag is processed by the APC for presentation to a responsive Th cell. If it is more efficient to process a single molecule for both helper and primary epitopes, than covalent linkage of B cells and T cell epitopes for intramolecular presentation of help would be recommended. If however, separate peptides containing either B or Th cell epitopes could be included within a single complex for the elicitation of intermolecular/intrastructural help, more antigenically diverse structures could be designed. This paper reports that it is possible to generate intermolecular/intrastructural help within an antigenic peptide-phospholipid (PL) complex. These peptide-PL complexes use well defined epitopes from Plasmodium falciparum as Ag. In addition to generating intrastructural help, we have shown that the Ir to these peptide-PL complexes is controlled by Ir genes and is similar to the Ir to the circumsporozoite protein of this pathogen.

Intranasal immunization with proteoliposomes protects against influenza.

Worldwide, influenza virus remains a serious disease which has successfully eluded numerous attempts to design a consistently effective vaccine. In part, these attempts have been thwarted because of a lack of basic understanding of the mechanisms which mediate protection and recovery from influenza infection. A better understanding of the roles of secretory antibody, serum antibody and cell mediated immunity vis-à-vis protection and recovery from influenza infection has allowed us more rationally to approach the design and administration of a vaccine for influenza. We have constructed a vaccine composed of glycoproteins from the envelopes of either influenza of Sendai virus embedded in a lipid bilayer (immunosomes) mimicking the presentation of the virus to the cells during natural infection. Intranasal immunization with these immunosomes induces an adequate systemic Ir compared with intramuscular immunization and a superior local IgA response. These animals were specifically protected from virus challenge.

Defining minimal requirements for antibody production to peptide antigens.

The role that individual determinants play in modulating the immune response of an organism to a pathogen is often obscured because of the complexity of the pathogen. In order to gain a better appreciation of the role of individual determinants in the immune response, a pathogen may be dissociated into smaller components, for example peptides representing specific epitopes. These isolated components are often poorly immunogenic and historically have required the use of adjuvants to stimulate antibody production. This report defines the minimal essential requirements for antibody production to a peptide in this system. These are the ability to stimulate both B- and T-helper lymphocytes, anchorage in a phospholipid complex and multivalency within the complex. When these conditions are met, no additional adjuvants are necessary. This procedure has allowed us to identify three distinct T-helper cell epitopes from HIV gp160. In addition, this information has been used to produce a simple, totally synthetic and highly immunogenic preparation for the production of antibodies to peptides.